SOME NOTES ON VIRUS RETENTION BY SAND

Transcription

1 J. Gen. App!. Microbiol., 23, (1977) SOME NOTES ON VIRUS RETENTION BY SAND MALAY CHAUDHURI, K. V. A. KOYA,1 AND N. SRIRAMULU2 Environmental Engineering Laboratory, Department of Civil Engineering, Indian Institute of Technology, Kanpur , India (Received September 12, 1977) Using bacterial virus MS2 against Escherichia coli, a systematic investigation was conducted to study virus retention by sand. Virus sorption on sand was influenced by water chemistry of the system. Both rate of sorption and sorptive capacity decreased with increase in the system ph in the range of However, the highest virus sorption was observed with a natural ground water high in calcium and magnesium, at ph 7.8. Results of the percolation column test and rapid sand filtration study showed that sand was effective in removing the model virus from percolating water as well as during filtration. It was observed that viruses were not immobilized or inactivated following their retention or removal by sand. Sand is an important environmental material in terms of water quality management and storage. Recharge of aquifers (natural recharge due to seepage of rainfall or runoff, or engineered recharge with surface water or treated waste water) is a significant element in the resource and quality management of water and waste water. Under favorable geological or topographical conditions, natural sand deposits may be converted to "intermittent sand filters" for filtering settled urban waste water or biologically treated effluents, and are quite effective in preparing effluents for reuse in industry, agriculture, and recreation, and for recharge to the ground (1). Sand is also used universally as a filter medium in engineered water purification installations. Therefore, in view of the present awareness about the occurrence, survival, and movement of viruses in the water environment, virus movement through sand becomes an important aspect of environmental quality management. Comprehensive reviews on sorption of viruses onto surfaces in soil and water (2) and virus survival in soil and ground water (3) are available. Several studies on virus movement by sand filtration in water and waste water treatment were undertaken. However, these studies were concerned mainly with the gross 1 Present address : Regional Engineering College, Calicut, India. 2 Present address : N. S. Engineering College, Hyderabad, India. 337

2 338 CHAUDHURI, KOYA, and SRIRAMULU VOL. 23 removal efficiencies without focussing much on the various system parameters affecting virus removal. It is rather difficult to correlate the available information due to widely varying experimental and field conditions. In general, viruses were often isolated from rapid sand-filtered water and waste water (4-6). However, marked improvement in virus removal was observed when viruses were suspended in lime flocculated secondary waste water effluent (7) or when the calcium and magnesium concentration of the waste water effluent was increased (8). Virus removal during sand filtration of water was observed to be considerably influenced by the flow rate by RoBECK (9) who observed 80-90% of the seeded poliovirus (1-6 x 104 PFU/ml) passing through cm (2 ft) of California dune sand at flow rates above 2.45 m/hr (1.0 gpm/ft2) while the same bed was found capable of removing 99% of the virus for a period of 98 days at m/day (20-40 gpd/ft2). Regarding virus movement through natural sand deposits, two conflicting reports are available. GRIGOR'EVA and GONCHARUK (10) reported virus penetration through undergound filtration beds in Russia, whereas filtration through a distance of 61 m (200 ft) through the m (8-12 ft) sand strata at the Santee Recreation Project removed all poliovirus from the reclaimed waste water (11). The above-mentioned studies indicate the importance of sand as a medium for virus removal from water. It is also apparent that there is a need to study the various system parameters which may affect virus retention by sand. The present research was initiated to investigate virus retention by sand using batch sorption tests and percolation, as well as filter column studies, taking into consideration the important variables that may affect virus sorption or retention. MATERIALS AND METHODS Bacterial virus MS2 (MS2 phage) against Escherichia co/i was selected as the model virus for this study because of its resemblance to human enteroviruses (polio, coxsackie, and echoviruses) in size, shape, and type of nucleic acid. Enteroviruses belong to the Picornavirinae subfamily of the Napoviridae family of the naked ribonucleic acid (RNA)-containing viruses with cubic symmetry whereas MS2 phage belongs to the Androphagovirinae subfamily of the same family (12). MS2 phage has a single-stranded RNA core surrounded by a lipid-free protein coat (isoelectric point, 3.9). It is polyhedron in structure having a diameter of 25 nm and molecular weight of 3.7x lo6g (13). Recently, a plant-scale study on virus removal by slow sand filtration showed practically identical results with poliovirus and MS2 phage (14). The initial stock suspension of MS2 phage and its host, Escherichia coli A-19, were obtained from the Environmental Engineering Laboratory, University of Illinois at Urbana-Champaign. Subsequent stock suspensions were prepared according to the procedure described elsewhere (I5). Soft agar technique of ADAMS (16) was adopted for virus enumeration, and virus concentrations are reported as plaque-forming units per milliliter (PFU/ml).

3 1977 Some Notes on Virus Retention by Sand 339 Beach sand from Malabar, Kerala, India, was used in the batch sorption tests as well as in the percolation column study. The sand was found to be free from any silt or clay. It was sieved through B. S. S. No. 200 (75 x 10-3 mm), autoclaved, and dried at 103 for 24 hr before use in the sorption experiments. For the percola~ tion column study, the sand sample as obtained was used. The sand used in the filtration experiments was filter sand (sphericity, 0.8) obtained from the local water treatment plant. It was washed several times in tap water, dried at 103 for 24 hr, and sieved to a geometric mean size of 0.5 mm before use. The reaction mixture in the sorption kinetic experiments consisted of 20 ml of buffer solution (0.2 M phosphate for ph 6.0 and 7.0, and 0.2 M borate for ph 8.4), 180 ml of distilled water including appropriate volume of the stock suspension of MS2 phage, and 2 g of sand. A ground water supply was also used. The source of the supply was a well located in the campus of the Indian Institute of Technology, Kanpur. A typical mineral analysis of the water was : ph 7.8; ionic strength 0.02; conductance ,usiemens/cm; bicarbonate alkalinity mg/liter as CaCO3; hardness 200 mg/liter as CaCO3; calcium mg/liter as CaCO3; magnesium mg/liter as MgCO3; chloride mg/liter; sulfate mg/liter; phosphate mg/liter; iron mg/liter; manganese mg/liter; silica mg/liter; sodium and potassium mg/ liter; and turbidity not detectable. For the sorption experiment with well water, 200 ml of this water was used instead of buffered distilled water. Bottles containing the reaction mixture were kept in a rotary shaker and samples withdrawn at predetermined time intervals, centrifuged at 5,900 x g for 10 min, and the supernatant assayed for virus. A control was always maintained to account for any loss or inactivation of the virus during the experiment. A Corning glass column, 60 x 4.6 cm (ID) (1.97 x 0.15 ft), provided with a perforated glass plate at the bottom was employed in the percolation column study. The column was filled to the desired height by carefully pouring sand from top and conditioned by saturating several times from above with distilled water prior to experimentation. The feed solution was prepared by adding the required volume of the MS2 phage stock suspension to the well water to give an input virus concentration of 1.05 x 106 PFU/ml. Application rate for the column was m/day (2.18 x 105 gpd) as determined by using a procedure similar to the one recommended for septic tank percolation system (17). The column was dosed intermittently four times a day and dosing was continued till virus breakthrough was observed. At the end of the percolation study, about 5 g of sand from the column were removed to study the fate of the retained viruses. The sand sample was mixed with 10 ml of 3 % beef extract and kept in cold for 12 hr with frequent agitation. Thereafter, it was centrifuged at 5,900 x g for 10 min and the supernatant assayed for virus. Filtration experiments at conventional rapid sand filtration rate, 4.9 m/hr (2 gpm/ft2), were carried out in a Corning glass column, 2.54 cm (1.0 in) ID, with

4 340 CHAUDHURI, KOYA, and SRIRAMULU VOL cm (17.6 in) depth of filter sand. A 60-liter plastic container equipped with a stirrer served as the overhead storage tank and provided a gravity filtration head of 250 cm (8.2 ft). Filtration rate was monitored through a flowmeter and kept constant during an experiment. The well water was used for preparing the influent water. Since in a natural system a fraction of the viruses may attach to the suspended particles constituting turbidity (virus in association with turbidity) whereas the other fraction may remain free (discrete viruses), filter runs were performed with the model virus suspended in turbidity-free (the well water adjusted to ph 6, 7, and 8 with HCl) as well as uncoagulated turbid water (the well water with an influent kaolinite turbidity of 20 formazin turbidity units (FTU)), and alumcoagulated water containing the model virus (the well water with initial kaolinite turbidity of 70 FTU clarified to 33 FTU by coagulation with 8 mg/liter aluminum sulfate followed by 30-min settling). Fig. 1. Kinetics of sorption of MS2 phage on beach sand., ph 6.0 (0.2 M phosphate); x, ph 7.0 (0.2 M phosphate); 0, ph 8.4 (0.2 M borate); o, ph 7.8 (well water). MS2 input conc. 1.1 x 10~ PFU/ml; Sand 10 g/liter; temp Sorption kinetic experiments were carried out at room temperature during winter (17-18 ) whereas the percolation and filtration studies were conducted during summer (30-36 ). All glassware used in the study were cleaned by soaking overnight in 0.3 % Teepol B-300 (Surfactants Private, Ltd., Bombay, India) to minimize virus sorption on glassware followed by rinsing in tap and distilled water. Sterilization of glassware were accomplished in a hot air oven at 180 for 2 hr or longer.

5 1977 Some Notes on Virus Retention by Sand 341 RESULTS AND DISCUSSION Kinetics of sorption of MS2 phage on beach sand (Fig. 1) show that bulk of the sorption occurs during the first min and a plateau is reached in about 80 min. Both rate of sorption and sorptive capacity decrease with increase in ph of the system and this observation is in accordance with the prediction of GERBA et al. (3). Decrease in virus sorption with increase in ph is presumably due to increased negativity of sand (18) as well as the virus (13) at higher ph. High virus sorption observed with the well water may be attributed to its high calcium (1 x 10-3 N) and magnesium (3 x 10-3 N) content. Presumably, presence of calcium and magnesium neutralizes or reduces the repulsive electrostatic potential between negatively charged viruses and sand particles, allows them to come close enough for intermolecular van der waals forces to interact and thus facilitates sorption (19). Alternatively, a cation bridging linking sand and virus may also be operative as was hypothesized for clay-virus systems (20). A sorption experiment conducted to study the sorption isotherm indicated conformity of virus sorption on sand to the Langmuir equation (21). Figure 2 shows the results of the percolation column study using 15 cm depth of beach sand and indicates high virus retention capacity of this sand. Test to study the fate of the retained viruses showed that they remained active following Bed volume Fig. 2. Virus breakthrough curve for beach sand. MS2 input conc x 108 PFU/ml; application rate m/day; ph 7.8 (well water); temp sorption. Apparently, virus retention or sorption on sand is a reversible process as was observed with clay-virus systems (20, 22) and cannot be considered a process of absolute immobilization or inactivation of the virus. It would appear that any process (e. g., changes in water quality in terms of cation or organic matter content) that results in a breakdown of virus association (attachment) with sand may result in their further movement. Data on virus removal by rapid sand filtration are plotted in Fig. 3. Removal

6 342 CHAUDHURI, KOYA, and SRIRAMULU VOL GI III Fig. 3. Virus removal by sand filtration. of discrete viruses at ph 6, 7, and 8 shows a trend similar to the sorption experiments (Fig. 1). Filter performance improves considerably when the model virus is present in association with kaolinite turbidity. Considering the calcium and magnesium content of the water it would appear that a major fraction of the viruses attached to the clay particles constituting turbidity (19,20) and better filter performance is presumably due to the fact that viruses attached to bigger clay particles ( ,um) are more efficiently removed in filtration due to better transport of the particles. The best filter performance is with the coagulated water as can be expected since alum coagulation reduces the electronegativity of the suspended particles, results in an increase in the suspended particle size, and the alum fiocs with entrapped viruses and clay particles are positively charged. All these facilitate particle transport and attachment during filtration. Similar high virus removals were reported by BERG et al. (7) during filtration of lime-flocculated secondary waste water effluent. It was also observed that the removed viruses were not immobilized or inactivated, and active viruses were detected in the filter backwash water. This would indicate that care must be taken in the disposal of filter backwash water. Input virus concentrations much higher than that expected in percolating or natural surface waters were employed in this study so that the percolant or filtrate could be assayed directly for virus without applying any virus concentration technique. Considering the estimated average enteric virus density in raw domestic

7 1977 Some Notes on Virus Retention by Sand 343 waste water to be 700 units/100 ml (23), 7-70 PFU/liter in treated waste water (24) and 10 PFU/liter in polluted surface waters (23), it seems from the present study that sand will probably retain or remove a large percentage of such viruses from percolating water or during water filtration. CONCLUSIONS The present study reemphasizes the importance of sand as an environmental material in retaining or removing viruses from percolating water and during water filtration. Virus sorption or retention by sand is significantly influenced by the water chemistry of the system, e. g., ph and bivalent cation (calcium and magnesium) concentration. The retained viruses are not permanently immobilized or inactivated and may migrate further through sand when conditions favoring desorption prevail. Care must be taken in the disposal of backwash water from rapid sand filters treating water containing pathogenic viruses. REFERENCES 1) G. M. FAIR, J. C. GEYER, and D. A. OKUN, "Water and Waste Water Engineering, Vol. 2, Water Purification and Waste Water Treatment and Disposal," John Wiley, New York (1968), p ) G. BITTON, Water Res., 9, 473 (1975). 3) C. P. GERBA, C. WALLIS, and J. L. MELNICK, J. Irrig. Drainage Div., Proc. Am. Soc. Civil Eng., 101, 157 (1975). 4) J. D. ISHERWOOD, Am. J. Public Health, 55, 1946 (1965). 5) R. LAAK and D. M. MCLEAN, Can. J. Hyg., 58,172 (1967). 6) I. NESTOR and L. COsTIN, J. Hyg. Epidemiol. Microbiol. Immunol., 15, 129 (1971). 7) G. BERG, R. B. DEAN, and D. R. DARLING, J. Am. Water Works Assoc., 60,193 (1968). 8) E. LEFLER and Y. KoTT, In Virus Survival in Water and Waste Water Systems, ed. by J. F. MALINA and B. P. SAGIK, University of Austin Press, Texas (1974), p ) G. G. RoBECK, N. A. CLARKE, and K. A. DOSTAL, J. Am. Water Works Assoc., 54, 1275 (1962). 10) L. B. GRIGOR'EVA and E. I. GONCHARUK, Hyg. Sanit. (USSR), 31,158 (1966). 11) J. C. MERRELL, Jr., W. F. JOPLING, R. F. BOTT, A. KATKO, and H. E. PINTLER, "The Santee Recreation Project, Santee, California; Final Report," No. WP-20-7, Federal Water Pollution Control Administration, Cincinnati (1967), p ) C. R. GOODHEART, "An Introduction to Virology," W. B. Saunders, Philadelphia (1969), p ) L. R. OVERBY, G. H. BARLOW, R. H. DOI, M. JACOB, and S. SPIEGELMAN, J. Bacteriol., 91, 442 (1966). 14) Fortyfifth Report of the Director of Water Examination; Metropolitan Water Board, London (1973), p ) M. CHAUDHURI and R. S. ENGELBRECHT, In Advances in Water Pollution Research, ed. by S. H. JENKINS, Pergamon Press, Oxford (1971), p. II-20/1. 16) M. H. ADAMS, "Bacteriophages," Interscience, New York (1959), p ) "Code of Practice for Design and Construction of Septic Tanks : Part I, Small Installations (First Revision)," IS-2470 (Part I)-1968, Indian Standards Institution, New Delhi (1969),

8 344 CHAUDHURI, KOYA, and SRIRAMULU VOL. 23 p ) N. SRIRAMULU, Investigation on Virus Removal by Filtration, Ph. D. Thesis, Indian Institute of Technology, Kanpur, India (1975). 19) S. A. SCHAUB, C. A. SoRBER, and G. W. TAYLOR, In Virus Survival in Water and Wastewater Systems, ed. by J. F. MALINA and B. P. SAGIK, University of Austin Press, Austin (1974), p ) G. F. CARLSON, F. E. WOODWARD, D. F. WENTWORTH, and 0. J. SPROUL, J. Water Pollut. Control Fed., 40, R89 (1968). 21) K. V. A. KOYA, Virus Retention by Selected Indian Soils, M. Tech. Thesis, Indian Institute of Technology, Kanpur, India (1975). 22) K. V. A. KoYA and M. CHAUDHURI, Frog. Water Tech., 9, 43, (1977). 23) N. A. CLARKS, G. BERG, P. W. KABLER, and S. L. CHANG, In Advances in Water Pollution Research, ed. by W. W. ECKENFELDER, Pergamon Press, Oxford (1964), Vol. 2, p ) 0. J. SPROUL, L. R. LAROCHELLE, D. F. WENTWORTH, and T. R. THORUP, Chem. Eng. Frog. Symp., 63,130 (1967).